Vibrational Modes of Ubiquinone in Cytochrome bo3 from Escherichia coli Identified by Fourier Transform Infrared Difference Spectroscopy and Specific 13C Lab
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- 作者：Petra Hellwig ; Tatsushi Mogi ; Farol L. Tomson ; Robert B. Gennis ; Jun Iwata ; Hideto Miyoshi ; and Werner Mä ; ntele
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：November 2, 1999
- 年：1999
- 卷：38
- 期：44
- 页码：14683 - 14689
- 全文大小：92K
- 年卷期：v.38,no.44(November 2, 1999)
- ISSN：1520-4995
文摘
In this study we present the infrared spectroscopic characterization of the bound ubiquinonein cytochrome bo3 from Escherichia coli. Electrochemically induced Fourier transform infrared (FTIR)difference spectra of 
UbiA (an oxidase devoid of bound ubiquinone) and UbiA reconstituted withubiquinone 2 and with isotopically labeled ubiquinone 2, where 13C was introduced either at the 1- or atthe 4-position of the ring (C=O groups), have been obtained. The vibrational modes of the quinone boundto the discussed high-affinity binding site (QH) are compared to those from the synthetic quinones insolution, leading to the assignment of the C=O modes to a split signal at 1658/1668 cm-1, with bothcarbonyls similarly contributing. The FTIR spectra of UbiA reconstituted with the labeled quinonesindicate an essentially symmetrical and weak hydrogen bonding of the two C=O groups from the neutralquinone with the protein and distinct conformations of the 2- and 3-methoxy groups. Perturbations of thevibrational modes of the 5-methyl side groups are discussed for a signal at 1452 cm-1. Only negligibleshifts of the aromatic ring modes can be reported for the reduced and the protonated form of the quinone.Alterations of the protein upon quinone binding are reflected in the electrochemically induced FTIRdifference spectra. In particular, difference signals at 1640-1633 cm-1 and 1700-1670 cm-1 indicatevariations of 
-sheet secondary structure elements and loops, bands at 1706 and 1678 cm-1 are tentativelyattributed to individual amino acids, and a difference signal a 1540 cm-1 is discussed to reflect an influenceon C=C modes of the porphyrin ring or on deprotonated propionate groups of the hemes. Further tentativeassignments are presented and discussed. The 13C labeling experiments allow the assignment of thevibrational modes of a bound ubiquinone 8 in the electrochemically induced FTIR difference spectra ofwild-type bo3.
UbiA (an oxidase devoid of bound ubiquinone) and UbiA reconstituted withubiquinone 2 and with isotopically labeled ubiquinone 2, where 13C was introduced either at the 1- or atthe 4-position of the ring (C=O groups), have been obtained. The vibrational modes of the quinone boundto the discussed high-affinity binding site (QH) are compared to those from the synthetic quinones insolution, leading to the assignment of the C=O modes to a split signal at 1658/1668 cm-1, with bothcarbonyls similarly contributing. The FTIR spectra of UbiA reconstituted with the labeled quinonesindicate an essentially symmetrical and weak hydrogen bonding of the two C=O groups from the neutralquinone with the protein and distinct conformations of the 2- and 3-methoxy groups. Perturbations of thevibrational modes of the 5-methyl side groups are discussed for a signal at 1452 cm-1. Only negligibleshifts of the aromatic ring modes can be reported for the reduced and the protonated form of the quinone.Alterations of the protein upon quinone binding are reflected in the electrochemically induced FTIRdifference spectra. In particular, difference signals at 1640-1633 cm-1 and 1700-1670 cm-1 indicatevariations of -sheet secondary structure elements and loops, bands at 1706 and 1678 cm-1 are tentativelyattributed to individual amino acids, and a difference signal a 1540 cm-1 is discussed to reflect an influenceon C=C modes of the porphyrin ring or on deprotonated propionate groups of the hemes. Further tentativeassignments are presented and discussed. The 13C labeling experiments allow the assignment of thevibrational modes of a bound ubiquinone 8 in the electrochemically induced FTIR difference spectra ofwild-type bo3.